The evaluation of HIV-1 vaccines will be facilitated by the availability of better assays for HIV-1-specific human CD4+ and CD8+ T cell immunity. Such assays should be robust, reproducible, and amenable to high throughput analysis of trans-shipped clinical specimens. We have developed and optimized a flow cytometry-based assay to detect specific CD4+ and CD8+ human T cell responses to cytomegalovirus (CMV) in cohorts of HIV-1-infected patients. This "cytokine flow cytometry" (CFC) assay has shown that the presence of CD4+ T cell responses against CMV is correlated with the absence or resolution of CMV-associated end organ disease. More recently, results from this assay have been shown to correlate with results obtained using major histocompatibility complex (MHC) Class I tetramers bearing specific epitopes of CMV. Since the CFC assay appears to reliably and specifically detect such antigen-specific responses, we have also developed related assays capable of detecting specific human CD4+ and CD8+ T cell responses to other AIDS-related opportunistic infections, including those caused by Mycobacterium tuberculosis, the Mycobacterium avium complex, cryptococcus, and human papilloma virus. We now propose to devise a similar CFC assay for the detection and quantitation of CD4+ and CD8+ human T cell responses against epitopes of HIV-1. Preliminary experiments indicate that it is possible to detect such responses against a whole virus vaccine preparation and against defined epitopes of HIV-1 Gag. To develop, optimize, and standardize this CFC assay, and to correlate it with results obtained using MHC Class I/HIV-1 peptide tetramers (Specific Aim 1), we have formed a collaboration with Becton Dickinson Biosciences, a leading flow cytometry reagent and equipment manufacturer and distributor. To further evaluate this CFC assay, we have made plans to analyze HIV-1-specific immune responses in patients who have been exposed to but not infected by HIV-1, who are in varying stages of HIV-1 disease progression, or who are HIV-1-infected nonprogressors (Specific Aim 2). Finally, to determine whether the results from this assay may facilitate the design and development of HIV-1 vaccines, we have established collaborations with several research groups that are testing HIV-1 vaccines (including The Immune Response Corporation, Chiron, VaxGen, and the laboratory of Dr. David Weiner at University of Pennsylvania) and we have made plans to analyze HIV-1-specific T cell responses in both HIV- 1-seropositive and -seronegative vaccinees (Specific Aim 3).